The Portugal captain and Ballon d'Or winner has missed Real's last three matches since limping off near the end of the Champions League quarter-final, first leg against Borussia Dortmund on April 2.
Ancelotti said on Friday there was a still a chance Ronaldo might recover for the clash at the Mestalla stadium in Valencia but confirmed at a news conference the player was unavailable.
"But we have a lot of confidence in this team," added the Italian, who declined to answer a question about who would start the Cup final in Ronaldo's absence.
"It has always reacted well to injuries and I think we can also do things very well tomorrow."
The latest edition of Spain's 'Clasico' between arch-rivals Real and Barca is their 228th meeting and a repeat of the 2011 Cup final, also held at the Mestalla, when Ronaldo's extra-time header secured a 1-0 win.
The Portuguese is top scorer in La Liga and the Champions League this season and Ancelotti will be desperate to have him back for the semi-finals of Europe's elite club competition later this month when they play holders Bayern Munich over two legs.
"We are losing a player with incredible quality. But it could be that Cristiano Ronaldo's absence will motivate the rest of the team even more," Ancelotti said.
"The matches we have played without him have gone very well, working harder, running more and working together more.
"Working as a unit is the of course the objective and that is what we have been training for.
"I don't think there will be any problem motivating the players tomorrow, they are all very motivated and very excited. I am seeing a lot of desire to play this final."
Ancelotti dismissed a suggestion Real could have avoided Ronaldo's injury if they had given him more rest.
"Cristiano did not rest because there was no need," he told reporters.
"There was no situation we saw that suggested he was tired and if he had been tired he would not have played.
"He has a problem that many players have at this stage of the season.
"We protect Cristiano in the same way we try to protect all the players as much as possible.
"It could be a risk playing him and we are not going to take that risk as there are very important games still to come."
Ancelotti's Barca counterpart Gerardo Martino also has injury worries and is waiting on the fitness of three of his recognised centre backs.
Captain Carles Puyol, who has barely featured this term due to a series of injuries, Gerard Pique and Marc Bartra were all included in the squad for the trip to Valencia and Martino said a decision would be taken on Wednesday whether they could play.
Barca's Spain playmaker Andres Iniesta said he expected Real to be just as much of a threat without Ronaldo.
"The team doesn't change whether he is playing or not," Iniesta told a news conference.
"We know how good they are and we know their potential. If Cristiano doesn't play somebody else at the same level will."
Barca, the record winners with 26 Cups, are looking for a morale-boosting victory after crashing out of the Champions League to Atletico Madrid on Wednesday.
The followed that up with a shock La Liga reverse at Granada on Saturday that left them four points behind leaders Atletico and one adrift of Real with five games left.
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